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1. Raccolta e preparazione <ol><li> Raccogliere il sangue venoso da pazienti con sospetto di sepsi e mettere in due BACTEC (BD, Sparks, MD) bottiglie di emocoltura, uno aerobici e anaerobici uno, insieme composto da un set di emocoltura. </li><li> Posizionare le bottiglie nel 9240 BACTEC strumento a 35 ° C. </li><li> Incubare fino a quando lo strumento viene attivato in allarme a causa della crescita di organismi in bottiglia emocoltura. </li><li> Rimuovere bottiglia dallo strumento emocoltura. </li></ol> 2. Preparazione dei reagenti prima della colorazione <ol><li> Preparare la soluzione di lavaggio con l&#39;aggiunta di 4 ml di soluzione di lavaggio 60x seguito da 240 ml di fresca acqua deionizzata o distillata direttamente al piatto colorazione. </li><li> Mettere il piatto nella colorazione ° 55 C a bagno d&#39;acqua calda. </li><li> Conservare il concentrato rimanente a 2-8 ° C. </li><li> Rimuovere il liquido di montaggio dal frigorifero e riscaldare a temperatura ambiente. </li></ol> 3. Colorazione di Gram <ol><li> Capovolgere delicatamente il flacone cultura del sangue. </li><li> Utilizzando un ago e la siringa o un apparato subcolture, rimuovere il sangue (circa 5 ml) dalla bottiglia e posto in una provetta sterile con tappo a vite. </li><li> Utilizzando una pipetta sterile, posizionare una goccia di sangue su un vetrino per microscopio. </li><li> Aria secca il vetrino e fissare in metanolo per 10 secondi. </li><li> Lasciare scorrere asciugare all&#39;aria. </li><li> Eseguire una colorazione di Gram sul film del sangue per determinare la morfologia dell&#39;organismo in crescita nella bottiglia emocoltura. </li><li> Utilizzando una lente immersione in olio, visualizzare diapositive. Cocchi Gram-positivi in ​​coppie e catene brevi osservati sul colorazione di Gram è più coerente con specie Enterococcus. </li><li> Questo risultato colorazione di Gram unità allora la scelta del proprio kit sonda PESCE PNA da utilizzare per l&#39;identificazione batterica. Questa cultura del sangue saranno testate in base alla E. faecalis / OE PESCE PNA macchia. </li></ol> 4. PESCE PNA Stain <ol><li> Mescolare la provetta contenente il sangue delicatamente agitando prima della preparazione striscio. </li><li> Porre una goccia di soluzione di fissaggio sul pozzo di un vetrino da microscopio PESCE PNA. </li><li> Trasferire 10 L o una piccola goccia dal tubo con la cultura del sangue positivo per la soluzione di fissaggio e mescolare delicatamente per emulsionare. </li><li> Fissare gli strisci di riscaldamento per 20 min. a 55 ° C su una piastra di riscaldamento. </li></ol> 5. Controllo Qualità del materiale Uno positivo e uno negativo vetrino di controllo di qualità deve essere provato con ogni lotto di vetrini per la colorazione. <ol><li> Usa E. faecalis / OE controllo Slides acquistato da AdvanDx a tale scopo o preparare strisci da colture liquide di ceppi di riferimento di E. faecalis e E. faecium come controllo positivo sia su diapositive separate o in comune in uno scivolo e Staphylococcus spp e Streptococcus spp come materiale di controllo negativo. </li></ol> I risultati QC dovrebbe essere in grado di monitorare le condizioni per esami appropriati, in particolare quelle che riguardano rigore ibridazione e la penetrazione della parete cellulare, dal momento che la metodologia PNA è progettato per ottimizzare la penetrazione della parete cellulare 6. Ibridazione <ol><li> Aggiungere una goccia di E. faecalis / OE PNA al pozzo sul vetrino con lo striscio. </li><li> Aggiungi coprioggetto. Evitare bolle d&#39;aria. </li><li> Incubare per 30 ± 5 min. a 55 ± 1 ° C sulla piastra di riscaldamento </li><li> A seguito di luogo di incubazione i vetrini in vettore scivolo </li></ol> 7. Lavaggio stringente <ol><li> Vettore scorrere immergere nella soluzione di lavaggio preriscaldato a 55 ° C e rimuovere con attenzione il coprioggetto. Spesso, il coprioggetto scivola delicatamente agitando il vetrino nella soluzione di lavaggio. Occasionalmente, il coprioggetto deve essere delicatamente spinto fuori con le pinze. </li><li> Incubare la soluzione di lavaggio per 30 ± 5 min. a 55 ± 1 ° C. </li><li> Rimuovere le diapositive dalla soluzione di lavaggio </li><li> Lasciare che il vetrino di aria secca </li></ol> 8. Montaggio <ol><li> Aggiungere una goccia di liquido di montaggio ad ogni diapositiva </li><li> Aggiungi coprioggetto. Evitare bolle d&#39;aria. </li><li> Esaminare il vetrino di un microscopio a fluorescenza entro 2 ore. </li><li> Non esporre le diapositive a luce solare diretta o altre fonti di luce forte come questo può portare alla estinzione di fluorescenza. </li></ol> 9. Interpretazione dei risultati Il microscopio a fluorescenza utilizzato per l&#39;esame diapositive devono essere equipaggiati con il filtro AdvanDx Dual Band e un obiettivo olio 60x o 100x. I vetrini QC devono essere esaminati prima di confermare che l&#39;ibridazione si è verificato. E. faecalis dovrebbe apparire come luminosi verdi fluorescenti cocchi in molteplici campi di vista e la E. faecium apparirà come rosso brillante cocchi. Non enterococchi vetrino di controllo dovrebbe apparire nonfluorescent. Dopo aver confermato il sistema dei controlli, le diapositive paziente può essere esaminato. In un primo momento il sangue film appare rossastro, ma il rosso e il verde cocchi sarà molto evidente. <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="Figura 1" /> Esempi rappresentativi Figura 1. Verdi-positivi E. faecalis (a sinistra), rosso positivo E. faecium (al centro), e negative (a destra) i risultati dei test. 10. Rappresentante Risultati Tre istituzioni sono stati inclusi in uno studio multicentrico studio clinico di valutazione del presente PESCE PNA macchia e confrontando un protocollo di 2,5 ore al ridotto 1,5 protocollo ora che è appena stato recensione. Un totale di 152 routine cocchi Gram-positivi in ​​coppie e catene (GPC) positivo bottiglie di emocoltura sono stati inclusi negli studi. Ci è stata del 100% (152/152) l&#39;accordo tra i risultati delle modificati e la procedura di test originale per E. faecalis / OE PNA PESCE: 41/41 E. faecalis; 33/33 altri enterococchi e 78/78 GPC altri (Tabella 1). Questo metodo di colorazione era squisita sensibilità e specificità nel corso di questo studio clinico (Tabella 2). <table border="1"><tbody><tr><td></td><td> Metodi di routine </td><td> E. faecalis / OE PNA PESCE Procedura Standard </td><td> E. faecalis / OE PNA procedimento FISH breve </td></tr><tr><td> E. faecalis </td><td> 41 </td><td> 41 (Verde Positivo) </td><td> 41 (Verde Positivo) </td></tr><tr><td> E. faecium </td><td> 27 </td><td> 27 (Red Positivo) </td><td> 27 (Red Positivo) </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2 (rosso positivo) </td><td> 2 (rosso positivo) </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2 (rosso positivo) </td><td> 2 (rosso positivo) </td></tr><tr><td> Altri Enterococcus spp. </td><td> 2 </td><td> 2 (rosso positivo) </td><td> 2 (rosso positivo) </td></tr><tr><td> S. pneumoniae </td><td> 17 </td><td> 17 (negativo) </td><td> 17 (negativo) </td></tr><tr><td> S. viridans </td><td> 33 </td><td> 33 (negativo) </td><td> 33 (negativo) </td></tr><tr><td> S. mitis </td><td> 1 </td><td> 1 (negativo) </td><td> 1 (negativo) </td></tr><tr><td> S. pyogenes </td><td> 3 </td><td> 3 (negativo) </td><td> 3 (negativo) </td></tr><tr><td> S. bovis </td><td> 2 </td><td> 2 (negativo) </td><td> 2 (negativo) </td></tr><tr><td> S. salivarius </td><td> 1 </td><td> 1 (negativo) </td><td> 1 (negativo) </td></tr><tr><td> S. sanguinis </td><td> 2 </td><td> 2 (negativo) </td><td> 2 (negativo) </td></tr><tr><td> S. agalagtae </td><td> 7 </td><td> 7 (negativo) </td><td> 7 (negativo) </td></tr><tr><td> Altri Streptococcus spp. </td><td> 7 </td><td> 7 (negativo) </td><td> 7 (negativo) </td></tr><tr><td> Abiotrophia spp. </td><td> 3 </td><td> 3 (negativo) </td><td> 3 (negativo) </td></tr><tr><td> Peptostrepococcus spp. </td><td> 2 </td><td> 2 (negativo) </td><td> 2 (negativo) </td></tr><tr><td> Totale </td><td> 152 </td><td> 152/152 100% di accordo </td><td> 152/152 100% di accordo </td></tr></tbody></table> Elenco Tabella 1. Dei batteri testati utilizzando sia lo standard (2.5hr) e rapido (1,5 h) protocollo. <table border="1"><tbody><tr><td> Sensibilità E.faecalis </td><td> Sensibilità Altri Enterococcus spp. </td><td> Specificità </td></tr><tr><td> 100% (41/41) 95% CI (93,0-100) </td><td> 100% (33/33) 33/33 95% CI (91,3-100) </td><td> 100% (78/78) 95% CI (96,2-100) </td></tr></tbody></table> Tabella 2. Sensibilità e Specificità del 1,5 ore protocollo FISH PNA.

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

La produzione di questo video-articolo è stato sponsorizzato da AdvanDx. Benjamin Crystal è un dipendente di AdvanDx, che rende gli strumenti e reagenti utilizzati in questo articolo.

La E. faecalis / OE saggio FISH PNA per enterococco può fornire l&#39;identificazione 2-3 giorni prima di metodi di coltura tradizionali. La procedura abbreviata per il test (1,5 hourr) fornisce una rapida identificazione che può essere utilizzato per un migliore approccio alla terapia antimicrobica e la prognosi del paziente. Uno studio a sostegno di questa è stata eseguita da Forrest, et al. (6). Oltre due anni consecutivi a partire dal 2005 il laboratorio di microbiologia identificato cocchi Gram-positivi in coppie e catene in crescita in bottiglie di emocoltura con metodi microbiologici tradizionali e nel 2006, aggiungendo la E. faecalis / OE PNA FISH. Inoltre, un algoritmo di trattamento sviluppato dal team istituzioni antimicrobici (AMT) di utilizzare efficacemente i dati PESCE ANP generati dal laboratorio in modo tempestivo. L&#39;outcome primario valutato era giunto il momento di emocoltura attingere per l&#39;attuazione di un&#39;efficace terapia antibiotica prima e dopo PESCE PNA è stato istituito nel flusso di lavoro dei laboratori. Gravità della malattia, la posizione del paziente e la terapia antimicrobica empirica sono stati misurati. Un totale di 224 pazienti con batteriemia enterococciche acquisite in ospedale sono stati valutati con 129 nel periodo pre-intervento e 95 nel periodo PESCE PNA. PESCE PNA identificato E. faecalis 3 giorni prima di culture tradizionali (1,1 vs 4,1 giorni, p &lt;0,001). PESCE PNA identificato E. faecium in media 2,3 giorni prima (1,1 vs 3,4 giorni, p &lt;0,001) ed era associata a una riduzione statisticamente significativa nel tempo di iniziare la terapia efficace (1,3 vs 3,1 giorni, p &lt;0,001) e una diminuzione 30 giorni mortalità (26% vs 45%, p = 0,04). Questo gruppo ha concluso che la E. faecalis / OE saggio PESCE PNA in combinazione con un algoritmo di trattamento AMT ha portato nelle precedenti l&#39;inizio di una appropriata terapia antimicrobica empirica per i pazienti con acquisite in ospedale E. faecium batteriemia. <ol><li> I reagenti non devono essere utilizzati dopo la data di scadenza stampata sulle etichette e reagenti non deve essere modificato in alcun modo. Dovrebbero rimanere in frigorifero quando non in uso o che potrebbero perdere potenza. </li><li> Bottiglie cultura del sangue devono essere miscelati prima del campionamento. </li><li> E &#39;importante non permettere che la punta del flacone contagocce di toccare uno striscio di sangue, perché questo potrebbe causare la contaminazione crociata di materiale tra le diapositive, o causare la contaminazione del reagente. </li><li> Assicurarsi che il bagno d&#39;acqua e il blocco di riscaldamento sono mantenuti alla temperatura costante di 55 ° C. </li><li> Non utilizzare filtri diversi da quelli del filtro Dual Band o colori non sarà chiaramente discernere. </li><li> Non utilizzare vetrini da microscopio diversi preparati microscopici o ibridazione non si verificherà. </li><li> E &#39;importante che il microscopio sta funzionando correttamente. Assicurarsi che il bulbo microscopio è approvato entro il numero di ore di utilizzo. </li></ol><ul><li> Alcune rare le specie Enterococcus non vengono rilevati dalla sonda di PNA ibridare le specie Enterococcus altri, come ad esempio E. asinii, E.dispar, E. haemoperoxidus, E. cecorum, E. columbae, saccharolyticus E., E. solitarius ed E . sulfuree. </li><li> Moraviensis E. è identificato come E. faecalis a causa di similarità di sequenza. </li><li> Alcuni ceppi di Streptococcus anginosus produrre una fluorescenza verde falso positivo a causa di somiglianze sequenza. </li><li> VersaTREK bottiglie cultura del sangue non sono state valutate nel corso degli studi clinici. Le prestazioni per rilevare E. Enterococcus faecalis ed altre spp. con bottiglie di emocoltura VersaTREK è sconosciuta. </li><li> Falsi positivi autofluorescenza verde può verificarsi se uno standard FITC filtro viene utilizzato al posto del filtro Dual Band. </li><li> Risultati falsi negativi possono verificarsi raramente a causa della crescita misti o causa di un errore nella tecnica di analisi. </li><li> Il tipo e la condizione della strumentazione utilizzata influenzerà l&#39;aspetto visivo dell&#39;immagine ottenuta. La fluorescenza può varydue al tipo di microscopio impiegato, della fonte di luce e il livello di rRNA nelle cellule </li><li> Isolamento su terreni solidi è necessario per differenziare la crescita mescolato con altri organismi e di identificare emocolture positive ottenendo un risultato negativo. </li><li> Il prodotto non è stato convalidato con campioni diversi da emocolture. </li></ul>

Reagenti E. faecalis / OE PESCE PNA è costituito dai seguenti componenti del kit: <ul><li> Soluzione Soluzione fissazione Fixation 3 ml PBS con un detergente. </li><li> E. faecalis / OE PNA E. faecalis / OE PNA 1,5 ml sonde di PNA in ibridazione. soluzione. Contiene il 30% formammide. </li><li> Lavare 60x 60x soluzione di soluzione di lavaggio 50 mL Tris-salina tamponata con detergente </li><li> Media montaggio Mezzo di montaggio 3 inibitore mL photobleaching in glicerolo </li><li> Controllo di qualità diapositive </li><li> <a href="http://www.advandx.com">AdvanDX</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

Gli enterococchi sono una causa comune di batteriemia con E. faecalis è la specie predominante seguito da E. faecium. Perché la resistenza alla ampicillina e vancomicina in E. faecalis è ancora raro in confronto alla resistenza in E. faecium, lo sviluppo di test rapidi consentono la differenziazione tra le specie enterococchi è importante per la terapia appropriata e sorveglianza della resistenza. La E. faecalis OE saggio PESCE PNA (AdvanDx, Woburn, MA) utilizza specie-specifico acido nucleico del peptide (PNA) delle sonde in una fluorescenza in formato ibridazione in situ ed offre un tempo per i risultati di 1,5 ore e il potenziale di fornire informazioni importanti per le specie-specifiche trattamento. Studi multicentrici sono stati condotti per valutare le prestazioni del 1,5 ore E. faecalis / OE procedimento FISH PNA rispetto alla originaria procedura di test 2,5 ore e di batteriologia metodi standard per l&#39;identificazione degli enterococchi direttamente da una bottiglia positiva cultura del sangue.

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Un protocollo rapido per l&#39;identificazione diretta di Enterococcus faecalis ed Enterococcus altre specie da una emocoltura positiva con un Peptide Nucleic Acid test di ibridizzazione fluorescente in situ (FISH PNA).

Una procedura 1,5 ore per l&#39;identificazione di Enterococcus Specie Direttamente da emocolture

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

a-15-hour-procedure-for-identification-enterococcus-species-directly

Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.8K Views.  Cedars-Sinai Medical Cente. Un protocollo rapido per l'identificazione diretta di Enterococcus faecalis ed Enterococcus altre specie da una emocoltura positiva con un Peptide Nucleic Acid test di ibridizzazione fluorescente in situ (FISH PNA).

Video: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione del complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Ricerca

Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Un giorno Scheme flusso di lavoro per batterica Rilevazione dei patogeni e test di resistenza antimicrobica dalle emocolture

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generazione di culture Raft organotipiche da cheratinociti umani primari

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolamento di precursori delle cellule B di sottoinsiemi di sangue del cordone ombelicale

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Totale Estrazione di proteine ​​e 2-D elettroforesi su gel Metodi per<em&gt; Burkholderia</em&gt; Specie

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedure per l&#39;identificazione Infettive prioni dopo il passaggio attraverso l&#39;apparato digerente di una specie aviaria

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Rapida identificazione dei batteri Gram-negativi dal sangue brodo di coltura Uso MALDI-TOF

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparazione di un Sangue cultura pellet per il Rapid batterica identificazione e test di sensibilità agli antibiotici

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...